Construction and application of novel feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases by engineering the N-terminal domain forl-phenylalanine synthesis

Abstract

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHP synthase) encoded by aroF is the first enzyme of the shikimate pathway. In the present study, an AroF variant with a deficiency in residue Ile11 (named AroF*) was shown to be insensitive to l-tyrosine. According to three-dimensional structure analysis, nine AroF variants were constructed with truncation of different N-terminal fragments, and overexpression of the variants AroF(Δ(1-9)) , AroF(Δ(1-10)) , AroF(Δ(1-12)) and, in particular, AroF(Δ(1-11)) significantly increased the accumulation of l-phenylalanine (l-Phe). However, the AroG and AroH variants with similar truncations of the N-terminal fragments decreased the production of l-Phe. By co-overexpressing AroF(Δ(1-11)) and PheA(fbr) , the production of l-Phe was increased from 2.36 ± 0.07 g L(-1) (co-overexpression of the wild-type AroF and PheA(fbr) ) to 4.29 ± 0.06 g L(-1) . The novel variant AroF(Δ(1-11)) showed great potential for the production of aromatic amino acids and their derivatives.

Advanced Biotechnology: Metabolically Engineered Cells for the Bio‐Based Production of Chemicals and Fuels, Materials, and Health‐Care Products

“…Further improvement can be expected from the current achievements in strain engineering 63b. 92b, 153 The collected expertise has additionally driven the production of related aromatic compounds, mainly with E. coli 91a. Shikimate, itself an intermediate of the aromatic amino acid metabolism, is probably the most popular and successful example thereof, with titers of 80 g L −1 achieved 25a.…”

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

“…The PPA amount was determined by measuring the absorbance at 640 nm [23]. l -phenylalanine was derivatized with o -phthaldialdehyde and measured by Agilent 1100 HPLC system (Agilent, Palo Alto, CA) equipped with a reversed-phase column (Zorbax Eclipse-AAA) at a column temperature of 30°C and UV detector at 338 nm [24]. Biomass concentration was assessed spectrophotometrically (UV-2450 PC; Shimadzu Co., Kyoto, Japan) at an optical density of 600.…”

Analysis of acetohydroxyacid synthase variants from branched-chain amino acids-producing strains and their effects on the synthesis of branched-chain amino acids in Corynebacterium glutamicum

“…The removal of protein in supernatant was performed by trichloroacetic acid (30 g/l) and filtered through a membrane (pore size = 0.22 lm). Amino acids were precolumn derivatized by ophthaldialdehyde (OPA) and then analyzed on an Agilent 1100 HPLC system (Agilent Technologies) equipped with a reversephase column (Zorbax Eclipse-AAA) and an UV detector at 338 nm according to the published procedure [21,22].…”